Advantages and Limitations of Cell Culture Models in Pediatric Drug Development

Peter C. Adamson, M.D.The Children’s Hospital of Philadelphia

Clonogenic Assay

Primary Bioassay of Human Tumor Stem Cells* Tumor stem cells are cell

renewal source and serve as seed of metastatic spread

Cytotoxicity in clonogenic assay proportional to cytotoxicity in vivo

*Hamburger AW, Salmon SE. Science, 197 (4302) 461-463; 1977.

Tritiated Thymidine Incorporation

3H-TdR measures cells in S-phase Quantifies cell number as cpm

Historical in vitro Assays

Clonogenic Assay Labor intensive Not readily amenable

to high throughput

3H-TdR Limitations of using

radioactivity Non-clonogenic method

Non-clonogenic Assays

MTT Assay Rapid colorimetric assay for cellular

growth and survival: application to proliferation and cytotoxcity assays*

*Mossman T. J Immunol Meth 1983;65:55-63.

N

N N

N

S

NCH3

CH3

+

C

N

NH

N N

S

NCH3

CH3

SuccinateDehydrogenase

MTT Formazan

Leukemia NSCLC Small Cell Colon CNS Melanoma Ovarian Renal

NCI 60 Cell Line Screen

Non-Clonogenic Assays MTT XTT SRB Trypan Blue DiscAssay FDA TACs Hoechst

WST-1 Acid Phosphatase DIMScan MTS Brd-U Luminescent-ATP

Non-Clonogenic Assays

Non-clonogenic assay ≈

Clonogenic assay ≈

Viable cell number ≈

In vivo cell growth ≈

Tumor growth in patient

Use of Cell Culture Models

Drug discovery Cellular pharmacology Study mechanism of action Study drug resistance As pediatric tumor models

Drug activity Dose (concentration)-schedule dependence Drug combinations

Limitations of Cell Culture Models Cell lines undergo transformation to allow for in

vitro growth Drugs may require metabolic activation or have

active metabolites Potential differences in drug exposure

Protein binding Drug disposition not modeled

Differences in tumor micro-environment Lack of vascularization Hypoxia

Other limitations…

Advantages of Cell Culture Models Not labor intensive Relatively low cost Moderate throughput capabilities Ability to study multiple cell lines Ability to study multiple combinations of drugs

Only system that mathematically determines synergy, additivity, and antagonism

Example: Determination of Synergy Problems with the “addition” method

Drug A 25% cell kill Drug B 25% cell kill Drug A + Drug B > 50% cell kill - synergy?

It’s not that simple Drug A 70% cell kill Drug B 70% cell kill Drug A + Drug B = 140% cell kill?

Median Effect Model

Example: Activity in Pediatric Tumors

BMS 247550 is an analog of epothilone B that binds tubulin, stabilizes mictrotubules by inhibiting tubulin depolymerization, blocks mitosis and causes apoptosis.

BMS 247550 is cytotoxic in taxane resistant tumors and tumor cell lines expressing the multidrug resistance phenotype (MDR).

Fox, Stover, Widemann, Fojo, Balis (AACR 2003)

BMS 247550: Pre-clinical Activity

IC50 (nM)

Cell Line BMS247550 Paclitaxel Vincristine Vinorelbine

HOS 8.6 ± 0.4 0.4 ± 0.03 44.7 ± 1.0 10.6 ± 0.4LD 8.2 ± 0.4 2.0 ± 0.2 5.0 ± 0.5 4.9 ± 3.1RD 16.8 ± 6.9 0.6 ± 0.03 38.4 ± 2.0 18.0 ± 0.6Daoy 9.2 ± 0.2 14.4 ± 0.5 14.9 ± 0.4 20.1 ± 1.1SK-N-AS 11.7 ± 1.3 8.6 ± 2.3 4.7 ± 0.4 0.8 ± 0.1G401 7.9 ± 0.1 6.8 ± 0.5 5.2 ± 0.1 1.9 ± 0.2

Daoy - - + + P S P S

SKNAS - - + + P S P S

LD - - + + P S P S

RD - - + + P S P S

HOS - - + + P S P S

G401 - - + + P S P S

46K

Fox, Stover, Widemann, Fojo, Balis (AACR 2003)

Example: Integration of New Agents

Induct

ion

Consolid

Mai

ntIM DDI

VCRVCRL-AspL-AspDNMDNMPDNPDN

IT MTXIT MTXIT Ara-CIT Ara-C

6-MP6-MPAra-CAra-CCTXCTXVCRVCR

Peg-ASPPeg-ASPC-XRTC-XRTIT MTXIT MTX

6-MP6-MPIV-MTXIV-MTX

Peg-ASPPeg-ASPIT MTXIT MTX

VCRVCRL-AspL-AspDoxDoxCTXCTX6-TG6-TGAra-CAra-CDexDex

IT MTXIT MTX

6-MP6-MPMTXMTX

VCR/PDNVCR/PDNIT MTXIT MTX

IMDI

6-MP6-MPIV-MTXIV-MTX

Peg-ASPPeg-ASPIT MTXIT MTX

VCRVCRL-AspL-AspDoxDoxCTXCTX6-TG6-TGAra-CAra-CDexDex

IT MTXIT MTX

High-Risk ALL Therapy

506U? ?

Asparaginase + 506U

0

20

40

60

80

100

0.001 0.01 0.1 1 10 100

MOLT4

Nelarabine (µM)

0

20

40

60

80

100

0.001 0.01 0.1 1 10 100

CEM

Nelarabine (µM)

Nelarabine --> Asn [-]

Asn [-] --> Nelarabine

% S

urv

ival

Jayaprakash, Adamson, Lampkin, Berg, Balis, Fox (AACR 2004)

Perspectives on Cell Culture Models

In vitro models are a cost efficient method to search for activity, but mechanistic based approaches likely will have higher yield

In vitro models can further our understanding of drug action in pediatric tumors

Moderate throughput is advantageous, especially when studying drug combinations

Perspectives on Cell Culture Models

For most cytotoxic agents, if it does not work in vitro, it will not work in vivo

If it takes supra-pharmacologic concentrations in vitro to have an effect, it will likely not fare well in vivo

If it works well in vitro, there is a reasonable likelihood that it will do absolutely nothing in vivo